Recombinant Desulfovibrio vulgaris UDP-3-O-[3-hydroxymyristoyl] N-acetylglucosamine deacetylase (lpxC)

What is the biological function of LpxC in Desulfovibrio vulgaris?

LpxC (UDP-3-O-[3-hydroxymyristoyl] N-acetylglucosamine deacetylase) in Desulfovibrio vulgaris catalyzes the committed step in the biosynthesis of lipid A, which forms the membrane anchor of lipopolysaccharide (LPS) in the outer leaflet of the Gram-negative bacterial outer membrane . This enzymatic activity is essential for bacterial cell viability, as lipid A provides structural integrity to the outer membrane .

In Desulfovibrio vulgaris specifically, the lpxC gene is identified as DVU2917 and annotated as a UDP-3-O-acyl-N-acetylglucosamine deacetylase . While the enzyme plays the same fundamental role across Gram-negative bacteria, studying its specific characteristics in D. vulgaris is particularly relevant given this organism's association with inflammatory conditions such as ulcerative colitis .

The deacetylase reaction specifically removes the N-acetyl group from UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine in the lipid A biosynthetic pathway. This reaction represents a critical control point in LPS production, which is essential for bacterial survival.

How does the structure of Desulfovibrio vulgaris LpxC compare to LpxC from other bacterial species?

While the specific crystal structure of Desulfovibrio vulgaris LpxC has not been fully detailed in the provided search results, insights can be drawn from structural studies of homologous LpxC enzymes such as the one from Aquifex aeolicus. The LpxC structure represents a previously unobserved α+β fold topology that likely evolved through primordial gene duplication and fusion .

Key structural features include:

• Two domains connected by a 16-residue linker, with each domain containing a five-stranded β-sheet and two principal α-helices

• An active site located at the interface between the two domains, flanked by two smaller subdomains: a βββ subdomain and a βαβ subdomain

• A catalytic zinc ion positioned at the base of an active site cleft, adjacent to a hydrophobic tunnel that accommodates fatty acid substrates

• A unique zinc-binding motif distinct from other zinc metalloproteases

These structural characteristics are likely conserved in D. vulgaris LpxC, though species-specific variations may exist that could influence substrate specificity, inhibitor binding, or catalytic efficiency. Comparative structural analysis between D. vulgaris LpxC and homologs from other bacterial species would provide valuable insights into potential functional differences.

What expression systems are most effective for producing recombinant Desulfovibrio vulgaris LpxC?

Based on the methodology described for Pseudomonas aeruginosa LpxC (PaLpxC), successful expression of functional LpxC enzymes requires careful attention to several factors:

• Zinc supplementation: Maintaining zinc in the expression medium is crucial for proper protein folding and function, as demonstrated by improved thermal stability and crystallization of PaLpxC when zinc was present during expression .

• Protein quality assessment: Multiple analytical techniques should be employed to assess protein quality:

  • Thermal shift analysis (TSA) to evaluate protein stability and homogeneity

  • 1H-15N HSQC NMR to examine protein folding

  • Functional assays to verify enzymatic activity

• Metal content verification: A colorimetric assay using 4-(2-pyridylazo)-resorcinol (PAR) can be employed to determine zinc content and verify proper metalation of the enzyme .

For D. vulgaris LpxC specifically, an E. coli expression system with supplemental zinc in the growth medium would likely be effective, with optimization of expression temperature, induction conditions, and purification strategy to obtain properly folded, active enzyme. The protein should be carefully characterized to ensure proper zinc incorporation and structural integrity before use in experimental studies.

What are the optimal conditions for assaying Desulfovibrio vulgaris LpxC activity in vitro?

Based on established LpxC assay methodologies, the following conditions and approaches are recommended for assaying D. vulgaris LpxC activity:

• Assay options:

  • Fluorescamine-based assay: Measures the reaction of fluorescamine with the free amino group generated on the substrate by LpxC

  • LCMS assay: Quantifies the amount of product formed, particularly useful when compounds being tested interfere with fluorescence-based assays

  • Binding assays: Isothermal titration calorimetry (ITC) can be used to determine binding affinities of inhibitors or substrates

• Buffer conditions:

  • 25 mM Hepes (pH 7.0)

  • 0.1 M NaCl

  • Zinc supplementation at 1:1 Zn2+:LpxC ratio

• Temperature: Assays are typically conducted at 30°C for optimal activity

• Controls:

  • Metal-free enzyme (prepared by EDTA treatment followed by extensive dialysis) to establish zinc-dependence

  • Inhibitor-free reactions to establish baseline activity

  • Substrate specificity controls (e.g., comparing UDP-3-O-(R-3-hydroxymyristoyl)-GlcNAc vs. UDP-GlcNAc) to verify enzyme specificity

When reporting activity data, results should be presented as the geometric mean of at least two determinations to ensure reliability .

How can researchers effectively produce and purify the enzyme's substrate UDP-3-O-[3-hydroxymyristoyl]-N-acetylglucosamine?

While the search results don't provide specific methods for synthesizing the substrate, a general approach based on established protocols would include:

• Chemical synthesis route:

  • Starting with UDP-N-acetylglucosamine as the base material

  • Selective acylation at the 3-O position with R-3-hydroxymyristoyl group

  • Protection and deprotection strategies to ensure regiospecific modification

  • Purification by HPLC to obtain the final substrate

• Enzymatic synthesis approach:

  • Using the upstream enzymes in the lipid A biosynthetic pathway (LpxA and others)

  • Incubating UDP-N-acetylglucosamine with purified LpxA and acyl-ACP donor

  • Isolating the product by chromatographic methods

• Substrate verification:

  • Mass spectrometry to confirm molecular weight

  • NMR spectroscopy to verify structure and stereochemistry

  • Activity testing with well-characterized LpxC enzymes

Researchers should verify substrate purity and structure before use in kinetic studies with D. vulgaris LpxC to ensure reliable and reproducible results.

What techniques are most effective for studying the metal ion requirements of D. vulgaris LpxC?

Based on the methodologies described for other LpxC enzymes, the following techniques are recommended for investigating the metal ion requirements of D. vulgaris LpxC:

• Metal content analysis:

  • Plasma emission spectroscopy for direct measurement of zinc content

  • Colorimetric assays using 4-(2-pyridylazo)-resorcinol (PAR) to determine zinc concentrations

• Metal depletion and reconstitution:

  • Prepare metal-free enzyme by dialysis against 1.0 mM EDTA in appropriate buffer (e.g., 25 mM Hepes pH 7.0, 0.1 M NaCl)

  • Remove EDTA by extensive dialysis against metal-free buffer

  • Reconstitute with controlled amounts of different metal ions to assess specificity

• Activity correlation studies:

  • Measure enzyme activity as a function of metal ion concentration

  • Test activity with different divalent metal ions (Zn2+, Mn2+, Co2+, Ni2+, etc.)

  • Assess the effects of metal-chelating agents such as dipicolinic acid (DPA) and EDTA on enzyme activity

• Structural studies:

  • X-ray crystallography with anomalous scattering to locate metal ions

  • Extended X-ray absorption fine structure (EXAFS) analysis to determine metal coordination geometry

The data in Table 1 summarizes typical approaches for investigating metal dependency:

What are the structural determinants of substrate specificity in D. vulgaris LpxC, and how do they differ from other bacterial LpxC enzymes?

The substrate specificity of LpxC enzymes is largely determined by their ability to accommodate the 3-O-fatty acid substituent of the substrate. Based on structural studies of LpxC from Aquifex aeolicus, key structural elements likely influence substrate specificity in D. vulgaris LpxC:

• Hydrophobic tunnel: LpxC contains a hydrophobic tunnel adjacent to the catalytic zinc ion that accommodates the fatty acid portion of the substrate . The dimensions and amino acid composition of this tunnel significantly influence substrate preference.

• Substrate recognition: Kinetic studies with LpxC have shown that the ester-linked R-3-hydroxymyristoyl chain increases kcat/KM by approximately 5 × 10^6-fold compared to the substrate lacking this acyl chain (UDP-GlcNAc) . This dramatic enhancement demonstrates the critical importance of the acyl chain for substrate recognition.

• Active site architecture: The zinc-binding site and surrounding residues form a specific recognition pocket for the N-acetyl group that undergoes deacetylation .

For D. vulgaris LpxC specifically, sequence analysis and homology modeling would be necessary to identify potential differences in the residues lining the hydrophobic tunnel and active site that might influence substrate chain length preference or catalytic efficiency compared to other bacterial LpxC enzymes.

Comparative analysis of these structural features among LpxC enzymes from different bacterial species could reveal adaptations specific to D. vulgaris that might correlate with its ecological niche or physiological requirements.

How do inhibition mechanisms of D. vulgaris LpxC compare with those of LpxC from pathogenic bacteria, and what are the implications for antibiotic development?

LpxC inhibition strategies have been extensively studied due to the enzyme's potential as an antibiotic target. For D. vulgaris LpxC, several considerations are important:

• Inhibitor binding mechanisms:

  • Hydroxamate compounds are known LpxC inhibitors that target the zinc ion

  • Para-(benzoyl)-phenylalanine has been identified as a potential LpxC inhibitor with good binding affinity (XP Gscore of -10.35 kcal/mol)

  • Simple inhibitors targeting the hydrophobic tunnel can bind with micromolar affinity

• Structure-activity relationships:

  • Molecular dynamics simulations suggest stable binding of para-(benzoyl)-phenylalanine to LpxC

  • The optimization of binding to the hydrophobic tunnel is critical for inhibitor potency

  • Non-hydroxamate inhibitors have been developed through fragment-based discovery approaches

• Comparative inhibition analysis:

  • Isothermal titration calorimetry can be used to determine inhibitor binding affinities

  • Multiple functional assays, including fluorescamine-based and LCMS methods, can quantify inhibition potency

Table 2 summarizes reported inhibition data for various compounds against LpxC:

Understanding the inhibition mechanisms of D. vulgaris LpxC could provide insights into selective targeting of this enzyme in inflammatory bowel conditions where D. vulgaris overgrowth has been implicated , while minimizing disruption of beneficial gut microbiota.

What role might D. vulgaris LpxC play in the pathogenesis of ulcerative colitis, and what experimental approaches could test this hypothesis?

Recent research has established connections between Desulfovibrio vulgaris and inflammatory bowel conditions, particularly ulcerative colitis (UC). Several lines of evidence and experimental approaches are relevant to investigating the potential role of D. vulgaris LpxC in this context:

• Clinical observations:

  • D. vulgaris is enriched in fecal samples of UC patients, with abundance correlating with disease severity

  • Increased levels of Desulfovibrio spp. have been reported in the crypt mucous gel of UC patients

• Pathogenic mechanisms:

  • D. vulgaris produces H2S through dissimilatory sulfate reduction, which may contribute to gut inflammation

  • D. vulgaris flagellin (DVF) interacts with leucine-rich repeat containing 19 (LRRC19) receptors, inducing pro-inflammatory cytokine production

  • Administration of D. vulgaris or DVF exacerbates dextran sulfate sodium (DSS)-induced colitis in mouse models

• LpxC-specific hypotheses:

  • LpxC produces lipid A, a component of LPS known to be highly immunoreactive

  • Alterations in LpxC activity could affect LPS structure and immunogenicity

  • Inhibiting LpxC could potentially reduce D. vulgaris viability or alter its inflammatory properties

• Experimental approaches to test D. vulgaris LpxC involvement:

  a. Mouse model experiments:

  • Compare wildtype D. vulgaris with LpxC-mutant strains in DSS colitis models

  • Test specific LpxC inhibitors for effects on D. vulgaris-exacerbated colitis

  • Analyze lipid A structures produced by D. vulgaris in inflammatory vs. normal conditions

  b. Ex vivo studies:

  • Culture intestinal organoids with wildtype or LpxC-modified D. vulgaris

  • Measure inflammatory markers and epithelial barrier integrity

  • Assess immune cell recruitment and activation

  c. Clinical correlations:

  • Analyze D. vulgaris LpxC expression levels in UC patient samples

  • Characterize lipid A structures in UC patients with high D. vulgaris abundance

  • Examine genetic variations in D. vulgaris lpxC gene from UC patients

These approaches could help determine whether D. vulgaris LpxC represents a viable therapeutic target for reducing inflammation in UC, possibly through selective inhibition that would reduce D. vulgaris viability or alter its inflammatory properties without disrupting beneficial gut microbiota.

How does the catalytic mechanism of D. vulgaris LpxC compare with other zinc-dependent deacetylases, and what implications does this have for inhibitor design?

The catalytic mechanism of LpxC involves zinc-dependent deacetylation, similar to other zinc-dependent deacetylases, but with unique structural features that influence its function:

• Zinc coordination and catalytic site:

  • LpxC contains a catalytic zinc ion at the base of the active site cleft

  • The zinc-binding motif in LpxC is distinct from other zinc metalloproteases

  • The active site architecture is specialized for recognition of the UDP-sugar substrate with the critical 3-O-acyl chain

• Catalytic mechanism:

  • The zinc ion likely polarizes the carbonyl group of the N-acetyl substrate

  • A general base abstracts a proton from water to generate a nucleophile

  • The resulting hydroxide ion attacks the carbonyl carbon

  • The tetrahedral intermediate collapses, releasing the deacetylated product

• Unique aspects of LpxC catalysis:

  • The dramatic enhancement in catalytic efficiency with acylated substrates (5 × 10^6-fold increase in kcat/KM) suggests the acyl chain plays a crucial role in substrate positioning

  • The hydrophobic tunnel that accommodates the acyl chain likely contributes to transition state stabilization

• Implications for inhibitor design:

  • Effective inhibitors should engage both the zinc center and the hydrophobic tunnel

  • The unique zinc-binding motif of LpxC offers opportunities for selective inhibition compared to other zinc-dependent enzymes

  • Fragment-based approaches have proven successful in discovering non-hydroxamate LpxC inhibitors

  • Molecular dynamics simulations can predict stability of inhibitor binding, as demonstrated with para-(benzoyl)-phenylalanine

For D. vulgaris LpxC specifically, inhibitor design should consider any unique features of its active site and hydrophobic tunnel that might differ from better-characterized LpxC enzymes. Comparative molecular modeling and docking studies would be valuable for identifying potential D. vulgaris-specific inhibitor optimizations.